Fig 2: A genome-wide CRISPR screen identified ZNF251 whose knockdonw caused Olaparib resistance in a breast cancer lineA CRISPR screen in MDA-MB-436 BRCA1-mutated breast cancer cells uncovered ZNF251 genes whose loss-of-function confers olaparib resistance. A, B. Enrichment of specific sgRNAs that target each gene after 14 days of olaparib treatment and identification of top candidate genes. The x axis represents enriched genes, and the y axis represents sgRNA enrichment score, which was calculated using (sgRNA number from the reads)/(sgRNA number in the library)/log2 (average abundance). Arrow indicates ZNF251 gene. C. Top panel: T7EN1 assay analysis of specific sgRNA-mediated in/dels at ZNF251 locus in MDA-MB-436 cells. Bottom panel: Cell growth curve of parental (ZNF257 WT) and ZNF251 KD MDA-MB-436 breast cancer cells following treatment with olaparib. The results represent three independent experiments. D. Top panel: Sanger sequencing data of three ZNF257 KD clones. Bottom panel: Western blot analysis of wildtype (WT) and ZNF251KD clones 7–3 of MDA-MB-436 breast cancer cells. GADPH was used as a loading control. The abundance of ZNF257 bands relative to the corresponding GADPH bands was assessed densitometrically. E. Cell growth curve of WT and three ZNF251KD single clone breast cancer cells following treatment with olaparib. The results represent three independent experiments. F. ZNF251KD was constructed in HCC7937 (BRCA1+ or BRCA1-) lines and the resistance to olaparib was measured. The results represent three independent experiments. G, H. The effect of olaparib on the growth of wildtype and ZNF251KD MDA-MB-436 breast cancer cells xenografts in immune-deficient NOD/SCID/IL-2Rγ (NSG) mice was tested. The results represent three independent experiments.

Fig 3: DNA-PKcs inhibition increase the fork degradation of olaparib-treated ZNF251 KD cellDNA-PKcs inhibitor increased fork degradation in ZNF257KD cells. A Top: schematic representation of the protection of nascent DNA at stalled replication forks employing DNA fiber assay. Bottom: representative images of protected and degraded DNA fibers. B. Graph summarizing the quantification of ldU/CIdU ratio for n = 100 DNA fibers analyzed per sample for each experiment (Cells were treated either 5 μM olaparib and/or 8 μM Plk-75). The graph is representative of 2 independently performed experiments. Significance was calculated with the Mann-Whitney U-test, and bar indicated the median for each sample. ****P < 0.0001 differences between samples.